Methods of quantifying biomarkers

ABSTRACT

Methods are provided for simultaneously measuring hematocrit (hct) level and the concentration of a biomarker in a blood specimen. Thus, serum biomarker concentrations can be more accurately measured. The methods are particularly useful for newborn screening programs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/060,647 filed Jun. 11, 2008, the disclosure of which is incorporatedhere by reference in its entirety.

BACKGROUND

In the first 15 years of newborn screening using a dried blood specimen(Guthrie spot), the assays provided a semi-quantitative result that wasevaluated by a technician comparing the observed result with a series ofcontrols with known values. With the introduction of RIA testing forthyroid disease in 1978, however, it became necessary to know thehematocrit (hct) in order to calculate the amount of biomarker thyroxin(T4) in the specimen. A CDC conference that year addressed the issue,noting that “the hct had little or no effect on serum volume in a ⅛-inchpunch.” (Proceedings of a Conference on a National Model forStandardization of Neonatal Hypothyroid Screening Programs, Atlanta,Centers for Disease Control (1978).) Consequently, it was decided that avalue of 55% would be used in all calculations derived from Guthrie spotanalysis. Most reports, even continuing to the present, adjust the hctto 55% in materials prepared as standards and controls in the study. TheCDC has adjusted its standards and controls to a 55% hct since 1978.

Twenty-three years later, the CDC newborn screening unit, now a robustQC/QA program for newborn screening throughout the world, published areport, showing that the amount of serum in the Guthrie spot wasdirectly proportional to the hct of the infant (Mei, J V, et al., J.Nutr. 131:1631 S-1636S (2001)). The report further noted that as thevolume of blood applied to the special testing paper increased, theamount of serum in the center of the ½ inch circle was increased.

Subsequently, many authors have addressed this issue, most recently in2006, when Holub et al. showed that the hct of the blood applied to thespecial paper used for this purpose and the location of the ⅛ inch punchtaken for analysis were significantly different from 55% and couldproduce, separately and in combination, incorrect analytical results(Holub, M., et al., Clin. Chim. Acta 373:27-31 (2006)). Earlier in 1995,an attempt was made to correct for the varying hcts by measuring sodiumin a separate punch from the specimen (Arends, J., et al., Screening4:101-105 (1995)). The study demonstrated that, when using the acceptedcut-off for normal thyroxine (T4) of 10% with an adjustment applied forhct, seven of 17 positive cases would be detected that had beenclassified as normal without the adjustment. Analyzing a sample from aseparate punch can introduce variability, however.

Thus, there is a need for a method for estimating, from a single punch,a biomarker's concentration and the hct in a Guthrie sample.

SUMMARY

In one aspect, a method is provided for determining a concentration of abiomarker in the serum of a patient, the method comprising (a) obtaininga blood sample from a patient, (b) obtaining an extract from the bloodsample, (c) measuring a concentration of hemoglobin (Hb) in the extract,(d) calculating a hematocrit (hct) value for the blood sample from themeasured concentration of Hb, (e) measuring a concentration of abiomarker in the extract, and (f) determining a concentration of thebiomarker in the serum of the patient using the calculated hct value,volume of the blood sample, and the measured concentration of biomarkerin the extract.

In another aspect, a method is provided for calculating a concentrationof a biomarker in the serum of a patient, comprising (a) measuring aconcentration of Hb and a concentration of a biomarker in an extractobtained from a dried blood sample from a patient; (b) converting themeasured concentration of Hb to a hct value; and (c) calculating aconcentration of the biomarker in the serum of the patient using the hctvalue and the measured concentration of the biomarker in the extract.

In yet another aspect, a method is provided for arriving atconcentrations of one or more biomarkers in a patient's whole blood,comprising (a) obtaining a punch from a dried whole blood specimen of apatient, wherein the dried whole blood specimen is made from a wholeblood sample taken from the patient; (b) obtaining an eluent from thepunch; (c) measuring concentrations of one or more biomarkers and of Hbin the eluent or a diluent thereof; (d) estimating an hct fraction ofthe whole blood sample from the measured concentration of Hb; and (e)adjusting the measured concentrations of the one or more biomarkersbased on the estimated hct fraction to arrive at concentrations of theone or more biomarkers in the serum fraction of the patient's wholeblood.

In another aspect, a method for determining a concentration of Hb in asample, the method comprising: (a) contacting the sample with afluorogenic substrate for peroxidase and hydrogen peroxide, such thatwhen Hb is present a fluorescent product is produced; (b) determiningthe amount of the fluorescent product; and (c) calculating theconcentration of the Hb in the sample based upon the amount of thefluorescent product determined in (b).

In some embodiments, the biomarker is a metabolic biomarker, and issuitable for use in a newborn screening program. The biomarker also maybe selected from the group consisting of T4, TSH, and an amino acid. Inother embodiments, the measurement of Hb and/or the biomarker isconducted using an affinity-based assay, which can be an immunoassay. Insome aspects, the concentration of Hb and/or the biomarker is measuredusing a multiplexed immunoassay.

In another aspect, a universal assay buffer is provided which is usefulin carrying out a substantially simultaneous analysis of two or morebiomarkers. Thus, in some aspects, an assay buffer for the substantiallysimultaneous analysis of two or more biomarkers is also provided, thebuffer comprising, in an aqueous mixture: (i)tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl); (ii) sodiumchloride (NaCl); (iii) one or more emulsifiers; (iv) bovine serumalbumin (BSA); (v) polyethylene glycol (PEG); (vi) one or morepreservatives; (vii) bovine globulin; and (viii) a testosteronederivative. In preferred embodiments, the buffer is substantially freeof 8-anilino-1-naphthalenesulfonic acid or a salt thereof.

In some embodiments, the aqueous buffer comprises, in an aqueousmixture: (i) about 50 mM Tris-HCl; (ii) about 150 mM NaCl; (iii) about0.02% Tween 40; (iv) about 1% BSA; (v) about 0.5% polyethylene glycol;(vi) a preservative; (vii) about 0.05% bovine globulin; (viii) about 0.5mg/L danazol; and (ix) about 1 mg/L of a protease inhibitor; providedthat the assay buffer does not include about 0.5 mg or more per liter of8-anilino-1-naphthalenesulfonic acid or a salt thereof.

In yet another aspect, a method is provided for arriving atconcentrations of two or more biomarkers in a serum portion of a wholeblood sample taken from a patient, the method comprising: (a) obtainingone or more punches from a dried whole blood specimen of a patient, inwhich the dried whole blood specimen is made from a whole blood sampletaken from the patient; (b) obtaining an eluent from the one or morepunches using a universal buffer as an elution solvent; (c) measuring aconcentration of each of two or more biomarkers and of hemoglobin (Hb)in the eluent or a diluent thereof, provided that the measurement of theconcentration of each of the two or more biomarkers is carried outsubstantially simultaneously using a multiplexed affinity assay; (d)estimating a hematocrit (hct) value for the whole blood sample from theconcentration of Hb measured in step (c); (e) using the estimated hctvalue to adjust the concentration of each of the two or more biomarkersmeasured in step (c) to arrive at concentrations of two or morebiomarkers in a serum portion of the whole blood sample taken from thepatient.

In another aspect, a method is provided for screening a patient for twoor more disorders by using a multiplexed affinity assay to determine theconcentrations of two or more biomarkers in a serum portion of a wholeblood sample taken from the patient, the method comprising: (a)obtaining a punch from a dried whole blood specimen of a patient, inwhich the dried whole blood specimen is made from a whole blood sampletaken from the patient; (b) obtaining an eluent from the punch using auniversal buffer as an elution solvent; (c) measuring a concentration ofeach of two or more biomarkers and of total hemoglobin (Hb) in theeluent or a diluent thereof, provided that the measurement of theconcentration of each of the two or more biomarkers is carried outsubstantially simultaneously using a multiplexed affinity assay; (d)estimating a hematocrit (hct) value for the whole blood sample from theconcentration of total Hb measured in step (c); (e) using the estimatedhct value to adjust the concentration of each of the two or morebiomarkers measured in step (c) to arrive at adjusted concentrations ofthe two or more biomarkers in a serum portion of the whole blood sampletaken from the patient; and (f) using the adjusted concentrations of thetwo or more biomarkers to determine whether the patient suffers from twoor more disorders.

Other objects, features and advantages will become apparent from thefollowing detailed description. The detailed description and specificexamples are given for illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Further, the examples demonstrate the principle of the invention andcannot be expected to specifically illustrate the application of thisinvention to all the examples where it will be obviously useful to thoseskilled in the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically illustrates the linear correlation of Hbconcentration with hct fraction (in %) for arterial blood samples. FIG.1 is from Kokholm, G., Scand. J. Clin. Lab. Invest. Suppl. 203:75-86(1990).

FIG. 2 graphically shows the theoretical effect of the hct of a bloodsample on the serum concentrations of thyroxine (T4) and TSH. “FP”denotes false positive, while “FN” denotes false negative.

FIG. 3 graphically plots mean fluorescent intensity (MFI) as a functionof the Hb concentration (mg/mL) for a series of Hb standards. The MFIvalues were obtained using a Luminex system in which Hb antibodies werecovalently coupled to a single set of beads and used to measure theconcentration of Hb in a series of Hb standards.

FIG. 4 presents standard curves for thyroxin (T4) and thyrotropin (TSH)derived using separate Luminex bead sets for each biomarker, asdescribed in Example 4 for the congenital hypothyroidism (CH) validationassay. In each standard curve, mean fluorescent intensity (MFI) isplotted as a function of the biomarker concentration for a series ofstandards for the biomarker.

FIG. 5 presents the results of the analysis for thyroxin (T4) in the CHvalidation assay described in Example 5. The Figure presents dataobtained using the Luminex system for sample 1038, a residual newbornsample, in fifteen replicate plates, using the controls routinelyprovided by the Centers for Disease Control (CDC). The Luminex resultsare compared with results from the newborn screening program. For theLuminex analysis results, each well in the plates contained a mixture ofthe thyroxin and TSH assays developed separately and then combined foranalysis of a single specimen. “C1” refers to the low control from theCDC for thyroxine, while “C2” refers to the high control.

FIG. 6 presents the results of the analysis for TSH in the CH validationassay described in Example 5. The Figure presents data obtained usingthe Luminex system for sample 1038, in 15 replicate plates, using thecontrols routinely provided by the CDC. The Luminex results are comparedwith results from the newborn screening program. Each well in the platescontained a mixture of the thyroxin and TSH assays developed separatelyand then combined for analysis of a single specimen. “C1” refers to thelow control from the CDC for TSH, while “C2” refers to the high control.

FIG. 7 presents performance parameters for the thyroxin and TSH resultsobtained for the CH validation assay described in Example 5, using theLuminex assay. The performance parameters are presented using thethree-level CDC controls.

FIG. 8 presents performance parameters for the thyroxin and TSH Luminexassay results from the CH validation assay described in Example 5 andcompares them with results obtained by the newborn screening program.

FIG. 9 presents standard curves for the two biomarkers immunoreactivetrypsin isoforms 1 and 2 (IRT1 and IRT2) derived using separate Luminexbead sets for each biomarker, as described in Example 5 for the cysticfibrosis (CF) validation assay. In each standard curve, mean fluorescentintensity (MFI) is plotted as a function of the biomarker concentrationfor a series of standards for the biomarker.

FIG. 10 shows the performance of the combined IRT1/2 bead sets from theLuminex assay described in Example 5 for the cystic fibrosis (CF)validation assay. The Table compares the Luminex assay results withresults from the newborn screening program.

FIG. 11 presents a standard curve for the 17OHP biomarker, as describedin the congenital adrenal hyperplasia (CAH) validation assay describedin Example 5. The standard curve was derived using a Luminex bead setfor 17OHP. In the standard curve, mean fluorescent intensity (MFI) isplotted as a function of the 17OHP concentration, for a series of 17OHPstandards.

FIG. 12 graphically presents the results of a correlation study of the17OHP assay results obtained using the Luminex bead set assay and theassay currently used in newborn screening. In the Figure, valuesobtained using the Luminex assay (y axis) are plotted as a function ofthe corresponding values obtained using the currently used assay (xaxis).

FIG. 13 presents a table displaying the results of four specimensanalyzed by the Example 5 multiplex biomarker assay to identifycongenital hypothyroidism (CH), cystic fibrosis (CF), and congenitaladrenal hyperplasia (CAH), as described in Example 5. The Luminex datais compared to similar results obtained in the three separate assayscurrently used to screen newborns.

FIG. 14 presents a standard curve for use in determining hematocritusing a single Luminex bead set detecting total hemoglobin. (left side),as described in Example 5. Results are also presented for threeconstructed hematocrit levels, which are compared with results obtainedusing Drabkins Reagent.

FIG. 15 graphically compares the IRT1+IRT2 (IRT1-IRT2) screen negativeby the IRT1 and IRT2 assay with the IRT-R (reference IRT), as discussedin Example 6.

FIG. 16 graphically illustrates the population distribution of the 597study samples discussed in Example 6.

DETAILED DESCRIPTION

Methods have been developed for accurately determining the concentrationof one or more biomarkers in the serum of a patient by taking intoaccount the effect of the hematocrit (hct). The methods involvemeasuring the concentration of hemoglobin (Hb) in an extract of aspecimen of a subject's blood, which also is used to measure theconcentration of a biomarker. The Hb concentration is used to calculatea hct value for the specimen, which in turn is used to calculate thesubject's true level of the biomarker. As the inventive methods moreaccurately measure serum biomarker concentrations, they represent animprovement over prior diagnostic assays that assign a value for the hctand will greatly benefit programs such as newborn screening.

In the methods, an extract is obtained from a sample of a patient'sblood, and the concentration of Hb in the extract is measured. Themeasured Hb then is used to calculate a hct value for the blood sample.Meanwhile, the concentration of one or more biomarkers in the extract ismeasured. The hct value is then used to calculate the concentration ofthe biomarker in the patient's serum or plasma, or in the patient'swhole blood.

The term “patient” or “subject” as used herein refers either to a humanor to a non-human mammal. Examples of non-human mammals include, but arenot limited to, primates, farm animals such as horses, sheep, or cattle,and domestic animals such as dogs, cats and the like.

In one aspect, the methods are used to measure biomarker concentrationsin the blood of human patients, in particular, human infants less thanabout 6 months of age or, more typically, human infants one day to twoweeks old. In one embodiment, the methods are used to measureconcentrations of one or more biomarkers in the serum of human infantswithin one or two weeks of birth, e.g., to screen human newborns forinborn errors of metabolism. In another aspect, the methods can be usedin veterinary applications to measure the concentrations of one or morebiomarkers in the blood of non-human patients, including primates, ordomestic animals such as farm animals and pets, including dogs and cats.

The terms “plasma” and “serum” as used herein refer to the liquidcomponent of whole blood in which the blood cells are suspended. Bothplasma and serum are clear, yellowish fluids that contain proteins,salts, sugars, vitamins, waste products. “Plasma” refers to the liquidcomponent of blood before clotting has taken place, and which containsfibrinogen and other clotting elements. “Serum” denotes the liquidcomponent of blood that remains after clotting, which lacks clottingelements and does not clot.

The terms “blood” and “whole blood” as used herein refer to plasmacombined with blood cells (e.g., red blood cells, white blood cells, andplatelets).

Both dried samples of blood and non-dried samples can be used in themethods. Examples of a non-dried blood sample include, but are notlimited to, a liquid sample of blood such as a blood sample freshlyobtained from a patient. Examples of dried blood samples include, butare not limited to, Guthrie spots (dried blood spots on filter paper).

In one embodiment, dried blood samples are used. In one example, thedried blood sample is in the form of a Guthrie spot. Methods forpreparing Guthrie spots are known to those of skill in the art, e.g.,clinicians and those with training in the field of medicine. Typically,the dried blood on the Guthrie spot is analyzed by first obtaining a⅛-inch diameter disk (−3.2 mm in diameter) from the Guthrie spot (i.e.,the disk is punched from the Guthrie spot), and then extracting theblood on the disk with an elution buffer to obtain an extract. Thevolume of blood on each ⅛-inch disk is generally estimated to be about 3microliters.

In some embodiments, the methods employ Guthrie spots to measure theconcentrations of a series of biomarkers in the serum or plasma of humaninfants to detect inborn errors of metabolism and the like.

In some aspects, a liquid blood sample is used for testing, e.g., for“point of care” screening of newborns. In these embodiments, the amountof blood for use in each sample can readily be determined by one ofskill in the art. Typically, liquid blood sample volumes are larger,typically three to five milliliters, than those of dried blood samples.For example, in some embodiments, the volume of the blood sample that istested is equivalent to about 3 microliters, the volume of bloodgenerally estimated to be present on a ⅛ inch diameter disk punched froma dried blood Guthrie spot.

Once a patient sample is secured, an extract of the patient's blood isobtained. In some embodiments, the extract is taken from a dried bloodsample, e.g., a Guthrie spot, using an elution buffer. Extracts of driedblood from Guthrie spots are typically obtained by punching a ⅛ inchdiameter disk from the Guthrie spot and incubating the disk with about100 uL of elution buffer for about 30 minutes, sometimes withsonication. Extracts of liquid blood samples are obtained similarly, byincubating an appropriate volume of liquid blood, generally about 3-5milliliters, in an appropriate volume of elution buffer, optionally withagitation. After elution, the resulting extract typically is filtered toremove solid particles.

Examples of suitable elution buffers include, but are not limited to,PBS (phosphate buffered saline) optionally combined with one or moreemulsifiers (e.g., Tween-20 or another detergent) at various pH values,including those at or close to physiological pH (e.g., at pH 7.4).

For example, elution of dried blood or liquid blood samples can beperformed using a universal assay buffer, which, in some embodiments,comprises in an aqueous mixture: (i) tris(hydroxymethyl)aminomethanehydrochloride (Tris-HCl); (ii) sodium chloride (NaCl); (iii) one or moreemulsifiers; (iv) bovine serum albumin (BSA); (v) polyethylene glycol(PEG); (vi) one or more preservatives; (vii) bovine globulin; and (viii)a testosterone derivative. Preferably, the universal assay buffer issubstantially free of 8-anilino-1-naphthalenesulfonic acid or a saltthereof. Preferably, the Tris-HCl is present at a concentration fallingin the range of about 25 to about 75 mM, more preferably, about 50 mMand that the pH of the buffer is adjusted to a pH of less than about 8,preferably falling in the range of about 7 to about 8, more preferablyabout 7.8 and most preferably about 7.75. Moreover, the NaCl ispreferably present at a concentration falling in the range of about 100mM to about 200 mM, more preferably, about 150 mM.

In a particular embodiment, the one or more emulsifiers in the universalassay buffer may comprise any one or more detergents. Suitableemulsifiers/detergents include polyoxyethylene sorbitan monopalmitate(a/k/a Tween 40), polyoxyethylene sorbitan monooleate (a/k/a Tween 80),and the like or combinations thereof. Preferably, the Tween 40 is usedas the emulsifier and the chosen emulsifier is present at aconcentration falling in the range of about 0.001% to about 0.1%, morepreferably, about 0.02%. Preferably, the BSA is present at aconcentration of about 0.5% to about 2%, more preferably, about 1%.

The preferred universal assay buffer also comprises PEG, preferably PEG6000. The PEG may be present at a concentration falling in the range ofabout 0.1% to about 1%, preferably, about 0.5%. Likewise, a bovineglobulin is included, which may be present at a concentration falling inthe range of about 0.01% to about 0.1%, preferably, about 0.05%. Asdescribed above, the inventive universal assay buffer includes atestosterone derivative. Examples of suitable testosterone derivativesinclude, but are not limited to, 17α-ethynyltestosterone (ethisterone),17β-hydroxy-2,4,17α-pregnadien-20-yno[2,3-D] isoxazole (danazol),19-nor-17α-ethynyltestosterone (norethindrone), or combinations thereof.A preferred testosterone derivative comprises danazol, and the chosentestosterone derivative may be present at a concentration ranging fromabout 0.1 mg per liter to about 1 mg per liter, preferably, about 0.5 mgper liter.

In one embodiment, the universal assay buffer contains one or morepreservatives, which may preferably be selected from (but not limitedto) sodium azide present at a concentration ranging from about 0.01% toabout 0.1%, preferably, about 0.05%, albumin present at a concentrationranging from about 0.1% to about 2%, preferably, about 1%, or acombination thereof. What is more, a preferred universal assay bufferincludes a protease inhibitor, such as (but not limited to) aprotinin,which may be present at a concentration ranging from about 0.1 mg perliter to about 2 mg per liter, more preferably, about 1 mg per liter,tranexamic acid or a salt thereof, which may be present at aconcentration ranging from about 0.1 mg per liter to about 2 mg perliter, more preferably, about 1 mg per liter, or a combination thereof.

Accordingly, in some embodiments, the elution buffer is a universalassay buffer comprising (in a water mixture): (i) about 50 mM Tris-HCl;(ii) about 150 mM NaCl; (iii) about 0.02% Tween 40; (iv) about 1% BSA;(v) about 0.5% polyethylene glycol; (vi) a preservative; (vii) about0.05% bovine globulin; (viii) about 0.5 mg/L danazol; and (ix) about 1mg/L of a protease inhibitor; provided that the buffer does not includeabout 0.5 mg or more per liter of 8-anilino-1-naphthalenesulfonic acidor a salt thereof, if at all. In one embodiment, the preservativecomprises about 0.05% sodium azide. In another embodiment, the proteaseinhibitor comprises about 1 mg/L aprotinin. An in still anotherembodiment, the buffer has a pH falling in the range of about 7 to about8.

Once an extract is obtained from the blood sample, the extract is thenanalyzed to determine the concentration of Hb and biomarker(s). Anybiomarker can be measured. Examples include, but are not limited to,amino acids, acylcarnitines, hormones such as thyroxine and thyrotropin,See Holub et al., Clin. Chim. Acta 373:27-31 (2006), and Mei et al., J.Nutr. 131:1631 S-1636S (2001), which are hereby incorporated byreference. The concentrations of many of these biomarkers are measuredin newborn screening programs to determine the presence of inborn errorsof metabolism. See, e.g., the list of conditions tested in the newbornscreening program conducted by the New York State Department of Health(www.wadsworth.org/newborn/babhealth.htm).

Suitable methods for measuring the amount of Hb and biomarker(s) in theblood sample extract are known to those of skill in the art and include,but are not limited to, chromatographic methods, such as HPLC (highperformance liquid chromatography), tandem mass spectrometry, andaffinity-based methods, such as immunoassays. Methods for calculatingthe concentration of an analyte (e.g., Hb and biomarkers) frommeasurements of the amount of the analyte from each of these methods arealso well known in the art.

In some embodiments, the extract is analyzed using an immunoassay.Immunoassays are well known in the art and can be performed usingdifferent formats, including a competitive or non-competitive(“sandwich”) format, and other variations known in the art. In someembodiments, the immunoassay can be performed using either a competitiveor non-competitive (“sandwich”) format, or both formats. For example,when multiple biomarkers are measured, either a competitive format or asandwich format can be used, so that different biomarkers can bemeasured using different formats in the same assay. In some embodiments,one biomarker can be measured using a competitive immunoassay format,while a second biomarker in the same assay can be measured using asandwich-type immunoassay format. In other embodiments, the same assayformat is used for each biomarker analyzed. In one aspect, an array canbe used. One example is a reverse-phase protein microarray.

The detection antibody used in an immunoassay is labeled with adetectable label, such as an enzymatic or fluorescent label, or aradioisotope. Methods of detecting the labeled antibody are well knownin the art. Examples, include but are not limited to, colorimetric,chemiluminescent, radiometric, or fluorometric methods.

In some embodiments, the detection antibody is labeled with afluorescent probe, which is detected using fluorometric methods known inthe art. A wide range of fluorescent probes are commercially available(See, e.g., Invitrogen Corporation, Carlsbad, Calif.). Examples ofsuitable fluorescent probes include, but are not limited to,phycoerythrin, including phycoerythrin-streptavidin andphycoerythrin-avidin conjugates. In addition, methods and reagents forcoupling fluorescent probes to proteins, including antibodies, are wellknown in the art. See, for example, technical handbooks from InvitrogenCorporation (Carlsbad, Calif.) and Pierce (Thermo Fisher Scientific,Inc., Rockford, Ill.).

A variety of antibodies to biomarkers are also commercially available.See, for example, Sigma-Aldrich (St. Louis, Mo.), Invitrogen Corporation(Carlsbad, Calif.), and BD Biosciences (San Jose, Calif.). Antibodiessuitable for use in the disclosed methods can be chosen readily by thoseskilled in the art.

In some embodiments, the extract of the patient's blood sample is testedfor multiple biomarkers using a multiplex assay format. In one example,multiplexed immunoassays are used. Examples of multiplexed immunoassaysinclude, but are not limited to, immunoassay-based protein microarrays,tandem mass spectrometry, and flow cytometric techniques.

For example, the amounts of Hb and biomarkers in the patient's bloodsample can be measured using an assay system from Luminex Corporation(Austin, Tex.), such as XMAP. The assay system uses five-micronpolystyrene beads that have been impregnated with a precise ratio of twofluorescent dyes, creating 100 spectrally identifiable beads. Thesurface of these beads is coated with carboxyl terminals (an estimatedone million) which serve as the attachment point for the immunoassaythat is built on the beads. Using the principles of traditionalimmunoassay, a sandwich or competition assay is developed for the targetbiomarker. At the completion of the four-hour assay, the beads are runthrough a modified flow cytometer. Two lasers query the beads: one forits ID number; the second for the intensity of the phycoerythrin signalresulting from the immunoassay. Up to 100 beads for each biomarker arecounted, then averaged to record the MFI (mean fluorescent intensity)for that assay. Since multiple beads sets can be used simultaneously inthe assay, the benefits of multiplexing can be utilized, allowingmultiple biomarkers for a condition (e.g., thyroxine and thyrotropin forcongenital hypothyroidism) and/or multiple conditions to be analyzedsubstantially simultaneously. See Bellisario, R., et al., Early Hum.Dev. 64:21-25 (2001), and Bellisario, R., et al., Clin. Chem.46(9):1422-1424 (2000).

In some aspects, evaluating a sample for multiple biomarkers can improvethe accuracy of detecting whether a patient has, or may develop, aparticular disorder. For example, when evaluating a patient sample forcongenital hypothyroidism, a combination of biomarkers TSH and T4 can beused. Similarly, when testing for congenital adrenal hyperplasia, acombination of markers 17OHP and cortisol can be used. When measuringfor cystic fibrosis, a combination of markers IRT1 and IRT2 can be used.

In other aspects, a sample is evaluated for the presence of multiplebiomarkers to ascertain whether a patient has, or may develop, one ormore of a variety of disorders. For instance, a sample can be evaluatedfor biomarkers indicative of disorders such as sickle cell disease,sickle cell trait, human immunodeficiency virus, homocystinuria,hypermethioninemia, branched-chain ketonuria, phenylketonuria,tyrosinemia, carnitine-acylcarnitine translocase deficiency, carnitinepalmitoyltransferase I deficiency, carnitine palmitoyltransferase IIdeficiency, carnitine uptake defect, 2,4-dienoyl-CoA reductasedeficiency, long-chain hydroxyacyl-CoA dehydrogenase deficiency,medium-chain acyl-CoA dehydrogenase deficiency, medium-chainketoacyl-CoA thiolase deficiency, medium/short-chain hydroxyacyl-CoAdehydrogenase deficiency, mitochondrial trifunctional proteindeficiency, multiple acyl-CoA dehyrdogenase deficiency, short-chainacyl-CoA dehyrdogenase deficiency, very long-chain acyl-CoAdehydrogenase deficiency, cobalamin A,B cofactor deficiency, cobalaminC,D cofactor deficiency, glutaric acidemia type I,3-hydroxy-3-methylglutaryl-CoA lyase deficiency, isobutyryl-CoAdehydrogenase deficiency, isovaleric acidemia, malonic acidemia,2-methylbutyryl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoAcarboxylase deficiency, 3-methylglutaconic acidemia,2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency,methylmalonyl-CoA mutase deficiency, mitochondrial acetoacetyl-CoAthiolase deficiency, multiple carboxylase deficiency, propionicacidemia, argininemia, argininosuccinic acidemia, citrullinemia,hyperammonemia/hyperornithinemia/homocitrullinemia, biotinidasedeficiency, galactosemia or Krabbe disease.

It is important to note that in some cases, it is desirable that atleast one of the two or more biomarkers comprises a protein biomarker,which is not a cytokine What is more, the two or more non-cytokineprotein biomarkers may include, but are not limited to, thyroxine (T4),thyrotropin (TSH), 17-hydroxyprogesterone (17OHP), an isoform ofimmunoreactive trypsin (IRT1), an isoform of immunoreactive trypsin(IRT2), cell surface receptor (CD3), a cell surface receptor (CD45),thyroxine binding globulin (TBG), pancreatic associated protein (PAP),as well as combinations thereof. In select cases, the universal assaybuffer is useful for the substantially simultaneous analysis of two ormore biomarkers, including 17OHP and a steroid or a steroid derivative,including cortisol, androsteindione, and the like. In still other casesthe universal assay buffer is useful for the substantially simultaneousanalysis of two or more biomarkers, including CD3, CD45 and a cytokine(IL-7).

Thus, in some embodiments, the present method also encompasses a methodof arriving at concentrations of two or more biomarkers in a serumportion of a whole blood sample taken from a patient, the methodcomprising: (a) obtaining one or more punches (preferably a singlepunch) from a dried whole blood specimen of a patient, in which thedried whole blood specimen is made from a whole blood sample taken fromthe patient; (b) obtaining an eluent from the one or more punches usingan universal assay buffer as an elution solvent; (c) measuring aconcentration of each of two or more biomarkers and of hemoglobin (Hb)in the eluent or a diluent thereof, provided that the measurement of theconcentration of each of the two or more biomarkers is carried outsubstantially simultaneously using a multiplexed affinity assay; (d)estimating a hematocrit (hct) value for the whole blood sample from theconcentration of Hb measured in step (c); (e) using the estimated hctvalue to adjust the concentration of each of the two or more biomarkersmeasured in step (c) to arrive at concentrations of two or morebiomarkers in a serum portion of the whole blood sample taken from thepatient.

In particularly preferred embodiments of the method, the eluent in (b)is obtained from a single punch of the dried whole blood specimen in(a). In these embodiments, multiple biomarkers are assayed using aneluent obtained from a single punch (e.g., a single 3 mm punch) takenfrom a single dried blood specimen (e.g., Guthrie spot). Using eluentfrom a single punch reduces variability in the measurement of thebiomarker concentrations, which can occur when two or more punches areused from the same dried blood specimen (same Guthrie spot). Suchvariations are often be due to slight differences in biomarkerconcentration that occur when the blood sample is spotted onto thecenter of the filter paper used to make the Guthrie spot andsubsequently “spreads out” on the paper.

A suitable multiplexed affinity assay may be one comprising amultiplexed bead-based affinity assay, a multiplexed electroluminescenceaffinity assay, a multiplexed chemiluminescence affinity assay, and thelike, or combinations thereof. If desired the affinity assay may includean immunoassay. Preferably, at least one of the two or more biomarkerscomprises a protein biomarker. And more preferably still, at least oneof the two or more biomarkers comprises a protein biomarker, which isnot a cytokine.

In certain cases, a preferred method further comprises determining ifthe estimated hct value is aberrant. Such aberrant hct values may arise,for example, because the affected patient either suffered or issuffering from anemia. These possibilities should preferably beconsidered.

In some embodiments, a universal assay buffer is contemplated for use inthe multiplexed assay. In these embodiments, the universal assay buffermay comprise, in an aqueous mixture: (i) tris(hydroxymethyl)aminomethanehydrochloride (Tris-HCl); (ii) sodium chloride (NaCl); (iii) one or moreemulsifiers or detergents; (iv) bovine serum albumin (BSA); (v)polyethylene glycol (PEG); (vi) one or more preservatives; (vii) bovineglobulin; (viii) danazol; and (ix) a protease inhibitor; provided thatthe assay buffer does not include about 0.5 mg or higher per liter of8-anilino-1-naphthalenesulfonic acid (ANSA) or a salt thereof, if any.In certain cases, however, it may be desirable to also include (orreplace the danazol altogether) with as little as about 100 μg per literor so of ANSA. In another embodiment, the universal assay buffer (whichagain is also the eluent used to elute the two or more biomarkers and Hbfrom the one or more punches, preferably, a single punch) has a pH ofabout 7.8 and consists essentially of: (i) about 50 mM Tris-HCl; (ii)about 150 mM NaCl; (iii) about 0.02% Tween 40; (iv) about 1% BSA; (v)about 0.5% polyethylene glycol-6000; (vi) about 0.05% sodium azide;(vii) about 0.05% bovine globulin; (viii) about 0.5 mg/L danazol; and(ix) about 1 mg/L aprotinin. Preferably, at least one of the two or morebiomarkers is a non-cytokine biomarker. More preferably, thenon-cytokine biomarker includes, but is not limited to, T4, TSH, 17OHP,cortisol, androsteindione, IRT1, IRT2, CD3, CD45, TBG, PAP, orcombinations thereof.

It may also be advantageous to carry out the step of using the estimatedhct value to adjust the concentration of each of the two or morebiomarkers measured in step (c) above with the aid of a processor and/ora computer algorithm configured for such purpose. While the methoddescribed has many applications, one particularly desirable applicationrelates to the carrying out of the method as part of a newborn screeningprogram. Hence, a suitable patient may include, but is not limited to, ahuman infant.

The concentration of Hb in the extract of the patient's blood sample isalso determined and then used to estimate a hct value for the originalblood sample. As discussed above, using the hemoglobin (Hb)concentration to estimate a hct value for the blood sample allows forthe more accurate measurement of serum biomarker concentrations in thedisclosed methods.

Measurement of the Hb in the extract also provides additional advantagesto the disclosed methods. For example, measuring the extract of thepatient's blood sample for Hb allows for the identification ofindividual Hb variants. As a result, in addition to screening a bloodsample for various biomarkers, the present methods can be used to screenthe sample for the presence of one or more specific Hb variants that canbe used to identify certain blood disorders, such as sickle cell anemia,that are associated with the variants. Thus, in some embodiments, thepresent method can be used to determine whether a subject or patienthas, or may develop, a blood disorder. In some embodiments, the presentmethod can also be used to determine whether a particular Hb variant ispresent in the patient's or subject's blood sample, or can be used tomeasure the concentration of, or the presence of, a particular Hbvariant in the patient's or subject's blood sample.

Measurement of the concentration of Hb in the extract can serve also asa positive control for the assay by providing assurance that the extractis physically present in the assay apparatus (e.g., ruling out operatoror machine error) and that the assay chemistry has performed as expected(e.g., that all assay components were delivered to the sample). Forexample, measuring a low or non-existent Hb concentration for a samplewould be an indication that the assay is not performing properly for thesample, or that the sample was absent. Thus, in some embodiments, thepresent method can be used to provide an improved method (e.g., a morereproducible, reliable, and/or more accurate method) of determining theconcentration(s) of one or more biomarkers in a blood sample of apatient or subject, by measuring both the concentration of Hb, or one ormore Hb variants, and the concentration(s) of the one or morebiomarkers, in an eluent or diluent thereof of a sample of a patient'sblood.

The term “hematocrit” as used herein refers to the proportion of bloodvolume that is occupied by red blood cells. The hct may be estimated bycentrifuging heparinized blood in a capillary tube to separate the bloodinto layers, and dividing the volume of packed red blood cells by thetotal volume of the blood sample.

Hemoglobin (Hb) is measured as described above. In some embodiments ofthe method, Hb is measured using the same assay format and method ofdetection that is used for one or more of the biomarkers. For example,Hb and one or more biomarkers can be measured using flow-cytometry, suchas in an assay system from Luminex Corporation (Austin, Tex.), in which,for example, Hb and each of the biomarkers are detected using antibodiesspecific for Hb and each biomarker. In some of these embodiments, bothHb and biomarkers are measured using a fluorescent probe bound to thedetection antibody. Using the amount of Hb measured in the extract, a Hbconcentration is then calculated for the original blood sample.

In some of these embodiments, for example, the Hb concentration can bemeasured using a pan-hemoglobin antibody. In other embodiments, the Hbconcentration can be measured by measuring the concentrations ofindividual Hb variants to arrive at a total Hb concentration. Forexample, in some embodiments, individual antibodies to each of a seriesof Hb variants can be used to measure the concentration of total Hb,rather than one pan-hemoglobin antibody which detects all Hb variants.In some of these embodiments, the concentrations of four or moreindividual variants are measured to arrive at the concentration of totalHb. Typically, at least five individual Hb variants are measured.Examples of Hb variants include, but are not limited to, Hemoglobin A(Hb A), Hemoglobin F (Hb F), Hemoglobin S (Hb S), Hemoglobin E (Hb E),Hemoglobin C (Hb C), and Hemoglobin H (Hb H).

Measurement of one or more individual Hb variants not only allows forthe estimation of a hct value for the original blood sample, but alsomakes possible the identification of the type of Hb present in the bloodsample, which can be used to determine whether a patient has, or maydevelop, one or more blood disorders that are associated with specificHb variants, such as, for example, sickle cell anemia, thalassemia, andhemolytic anemia.

Thus, is some embodiments, the extract of the patient's blood sample istested for multiple biomarkers using a multiplex assay format, in whichthe concentrations of two or more biomarkers are measured (e.g.,concentrations of the biomarkers T4, TSH, IRT1, IRT2, and 17OHP), inaddition to measurement of the concentrations of four or more individualHb variants (e.g., Hb A, Hb F, Hb S, Hb E, and Hb C). Measurement ofthese biomarkers allows one to determine at the same time whether thepatient has, or may develop, one or more disorders associated with thebiomarkers as well as those disorders associated with one or more of theindividual Hb variants, such as sickle cell anemia, which is associatedwith Hb S.

In other embodiments, Hb is measured using an assay format, or a methodof detection, that is different from that used for one or more of thebiomarkers. For example, Hb can be measured using a separate assay. Forexample, Hb concentrations can be measured using an assay based on thepseudoperoxidase activity of heme. In this assay, the hemepseudoperoxidase activity catalyzes the conversion of a substrate to afluorescent product in the presence of hydrogen peroxide. Acceptablesubstrates include, but are not limited to AMPLEX RED or AMPLEX ULTRARED(Invitrogen Corporation, Carlsbad, Calif.). In the presence of aperoxidase, the substrate reacts with hydrogen peroxide in a 1:1stoichiometry to produce resorufin, a highly fluorescent product. SeeMOLECULAR PROBES handbook, Section 10.5 (Invitrogen Corporation,Carlsbad, Calif.). The fluorescent product in the assay is then measuredusing known fluorescence detection methods. For example, the resultingfluorescent product can be detected using fluorescent flow cytometry,such as the Luminex assay system described above. When the Luminex assaysystem is utilized, the fluorescent product (resorufin) can be detectedusing aptamer-conjugated microspheres that capture the fluorescentreaction product. Examples of suitable aptamers are disclosed in Asai etal., Nucl. Acids Res. (supplement 3):321-322 (2003).

Thus, in some embodiments, both biomarkers and Hb are measured using afluorescence-based detection system, such as the Luminex system, inwhich one or more biomarkers are measured using a fluorescent probebound to each biomarker-specific antibody, while Hb is measured usingthe pseudoperoxidase assay, as described above, in whichaptamer-conjugated microspheres capture the fluorescent product (e.g.,resorufin) produced by the pseudoperoxidase reaction of the heme in theHb.

After the Hb concentration is calculated, the calculation is convertedto an estimated hct value for the original blood sample using thefollowing equation, which is taken from Kokholm, G., Scand. J. Clin.Lab. Invest. Suppl. 203:75-86 (1990) (the contents of which is hereinincorporated by reference in its entirety):

xHct=0.0485+ctHb+0.0083

where “ctHb” represents Hb concentration (mm/L), and “xHct” representsthe corresponding hct value. The hct value is then used to calculate thevolume of serum in the original sample, which is used in the calculationof the concentration of the biomarker that is also measured from thesame extract of the same blood sample. This allows for a more accuratedetermination of serum biomarker concentrations. Plasma biomarkerconcentrations can be calculated in a similar fashion. Concentrations ofthe biomarkers are calculated using methods known in the art that areappropriate for the individual assay format and detection method used.

While the calculations of the Hb, hct and biomarker concentrations canbe estimated by hand, standard software typically provided withluminometers and the like can greatly facilitate the process.

EXAMPLES Example 1

Antibodies against the Hb molecule were used to create an assay on asingle set of beads to measure the Hb concentration in a Guthrie spot.An assay system from Luminex Corporation (Austin, Tex.) was used.

The standards shown in FIG. 3 were diluted 1:100 in assay buffer(phosphate buffered saline/0.2% gelatin). A 75 ul aliquot was applied toeach well of a filter assay plate (Millipore MABVN1250, MilliporeCorporation, Billerica, Mass.). 50 ul of human Hb labeled with biotinwas applied to each well. Luminex microspheres conjugated with a pan-Hbantibody (25 ul) were applied and incubated for 60 minutes with shakingat 37° C. The beads were washed three times in phosphate bufferedsaline/0.05% Tween 20. Streptavidin PE (phycoerythrin conjugated tostreptavidin) (100 ul, 4 ug/mL) was applied and incubated for 30 minuteswith shaking Beads were washed one time in phosphate bufferedsaline/tween 20, resuspended in Luminex sheath fluid and analyzed on theLuminex system.

Mean fluorescent intensity (MFI) was measured for each Hb standard andplotted as a function of the Hb concentration (mg/mL) in the standard,as shown in FIG. 3. Curve fitting software (LIQUICHIP, Qiagen Inc.,Valencia, Calif.) was used to fit the displacement curve.

As shown in FIG. 3, the assay is linear across the range of normal Hbconcentrations. A corresponding hct value can be obtained by convertingthe Hb concentration read from the plot (shown in FIG. 1) to itscorresponding hct value using the following equation:xHct=0.0485+ctHb+0.0083, where “ctHb” represents Hb concentration(mm/L), and “xHct” represents the corresponding hct value. See Kokholm,G., Scand. J. Clin. Lab. Invest. Suppl. 203:75-86 (1990), which isherein incorporated by reference.

Example 2

The pseudoperoxidase activity of Hb can be used as the basis of analternative assay for measuring Hb. In one aspect, the assay utilizesthe Luminex system (Luminex Corporation, Austin, Tex.), and uses asingle set of beads to measure the Hb concentration in a sample.

A series of Hb standards are prepared in sample buffer. Separately, aset of Luminex aptamer-conjugated microsphere beads is prepared bycovalently coupling an aptamer, for example 5′-CCCCCCGGGGGGGTGGGGGGG-3′,to the beads according to the manufacturer's instructions (LuminexCorporation, Austin, Tex.), or according to coupling procedures known inthe art.

The Hb standards are prepared in assay buffer (phosphate bufferedsaline/0.2% gelatin), and a 75 ul aliquot of each diluted standard isapplied to each well of a filter assay plate (Millipore MABVN1250,Millipore Corporation, Billerica, Mass.). Appropriate amounts of afluorogenic substrate, such as AMPLEX RED (Invitrogen Corporation,Carlsbad, Calif.), and hydrogen peroxide are added to each well andincubated. During incubation, the reaction mixture in each well isprotected from light.

The aptamer-conjugated beads (25 ul) are then applied to the wellscontaining the Hb standards and hydrogen peroxide, and incubated for 60minutes with shaking at 37° C. The beads are then washed three times inphosphate buffered saline/0.05% Tween 20, and resuspended in Luminexsheath fluid for analysis in the Luminex system.

Mean fluorescent intensity (MFI) is measured for each Hb standard andplotted as a function of the Hb concentration (mg/mL) in the standard.Curve fitting software (LIQUICHIP, Qiagen Inc., Valencia, Calif.) isused to fit the displacement curve.

Example 3

A sample of dried blood from a Guthrie spot is assayed for thyroxine(T₄) using two sets of fluorescent microsphere beads, one set to measurethe concentration of T₄ in the dried blood sample and a second set tomeasure the Hb concentration.

The assay consists of two microtiter wells as follows: The first wellcontains one set of microspheres to detect the quantity of T₄ present inthe Guthrie spot eluent according to the protocol outlined in Bellisarioet al. (“Simultaneous Measurement of Thyroxine and Thyrotropin fromNewborn Dried Blood-Spot Specimens Using a Multiplexed FluorescentMicrosphere Immunoassay,” Clin. Chem. 46(9):1422-1424 (2000), which ishereby incorporated by reference). The second well contains a second setof microspheres to quantify Hb using the same Guthrie spot eluentappropriately diluted (e.g., 1 part eluent:99 parts eluent buffer), asdescribed in Example 1 above. A competitive-inhibition assay is used formeasurement of T4, and a sandwich-capture assay format is used formeasuring Hb.

Briefly, T₄-BSA antigen and anti-Hb (anti-Hb) monoclonal or polyclonalcapture antibody are covalently coupled to two microsphere setsaccording to the manufacturer's instructions (Luminex Corporation,Austin, Tex.). Preferably, the polyclonal capture antibody is one with apan-Hb specificity recognizing Hbs A1, A2, F and S and other Hbvariants. Alternatively, multiple monoclonals to the various Hb variantscan be utilized. The anti-Hb monoclonal or polyclonal antibody (e.g.,100 ug), and the T4-BSA antigen (e.g., 25 ug) are separately covalentlyattached to the carboxylate groups of two distinct microsphere setsusing a two-step coupling method. In the first step, the microspheres(10⁷ microspheres) are activated with 0.25 mg of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.25 mgof N-hydroxysulfosuccinimide (Sulfo-NHS) in 0.5 mL of 0.1 mol/L sodiumphosphate buffer, pH 6.1, for 20 minutes at room temperature. Themicrospheres are centrifuged and resuspended in 0.5 mL ofphosphate-buffered saline (PBS), pH 7.4. After a second wash, eachprotein is covalently coupled to its microsphere set in 0.5 mL of PBS,pH 7.4, by incubation for 2 hours at room temperature. The coupledmicrospheres are stored in PBS (pH 7.4) containing 10 g/L bovine serumalbumin and 0.5 g/L sodium azide (storage buffer).

The anti-Hb monoclonal or polyclonal detection antibody and the anti-T4monoclonal detection antibody are biotinylated separately according tothe manufacturer's instructions. For example, each detection antibody isbiotinylated with 40 ug of Sulfo-NHS-LC-Biotin (from Pierce) in 100 uLof PBS, pH 7.4, for 30 minutes at room temperature. The biotinylatedantibodies are stored in PBS (pH 7.4) containing 10 g/L bovine serumalbumin and 0.5 g/L sodium azide (storage buffer).

Calibration curves are first constructed for T₄ and Hb using T₄ and Hbdried blood-spot calibrators and controls, which are prepared asdescribed in Reilly et al., Clin. Chem. 44(2):317-326 (1998), which ishereby incorporated by reference, and in Example 1 above. Anapproximately 3.2 mm (⅛ inch) diameter disk is punched from each driedblood-spot T₄ and Hb calibrator or control and eluted with 100 uL ofphosphate buffered saline, pH 7.4, containing 0.05% Tween-20 and 0.2%gelatin, by sonication at room temperature for 30 minutes. The volume ofblood per 3.2 mm disk is assumed to be about 3 microliters. The eluentis filtered in a 0.45 um centrifugal filter unit (Millipore Corporation,Billerica, Mass.).

For the assay, about 50 uL (−5000 microspheres) of the boundT₄-BSA-microsphere set and of the anti-Hb capture antibody-microsphereset are added to individual wells in a 96-well filter-bottom microtiterplate (Millipore Corporation, Billerica, Mass.), with each wellcontaining only one set of microspheres. The microspheres in each wellare washed with 200 uL of PBS (pH 7.4) containing 0.5 mL/L Tween 20(PBS-Tween).

For those wells containing T₄-BSA-bound microspheres, filteredblood-spot eluent (e.g., 50 uL) and 50 uL of biotinylated anti-T₄detection antibody (0.25 mg/L in storage buffer) are added to theT₄-BSA-bound microspheres and incubated at 37° C. for 30 minutes, withshaking. For those wells containing anti-Hb antibody-bound microspheres,the filtered blood-spot eluent is first diluted appropriately. Thediluted eluent (50 uL) and 50 uL of biotinylated anti-Hb detectionantibody (50 uL; 4 mg/L in storage buffer) then are added and incubatedwith the anti-Hb antibody-bound microspheres at 37° C. for 30 minutes,with shaking. After incubation, the microtiter plate is washed threetimes with 200 uL of PBS-Tween. Streptavidin R-phycoerythrin (InvitrogenCorporation (Carlsbad, Calif.)) (50 uL) is then added to each well andincubated with shaking at 37° C. for 15 minutes. A final wash isperformed, and the microspheres are resuspended in 100 uL of PBS-Tweenfor analysis and data collection using a Luminex¹⁰⁰ instrument (LuminexCorporation, Austin, Tex.) in multiplexed acquisition mode, gated toexclude microsphere multimers. The instrument calculates the robust meanand the median fluorescence intensity from 100 microspheres of each set.A calibration curve is then constructed for T₄ as described inBellisario et al., Clin. Chem. 46(9):1422-1424 (2000). A calibrationcurve for Hb is calculated using LIQUICHIP analysis software (QiagenInc., Valencia, Calif., USA). All T₄ and Hb concentrations on thecalibration curves are expressed in units of whole blood.

In another approach for measuring Hb, purified human Hb is biotinylatedand used in a competitive assay format. For example, a 75 ul aliquot ofthe dried blood spot eluent diluted 100-fold is applied to each well ofa filter assay plate (Millipore MABVN1250). 50 ul of human Hb labeledwith biotin is applied to each well. Luminex microspheres conjugatedwith a pan-Hb antibody (25 ul) are applied and incubated for 60 minuteswith shaking at 37° C. The beads are washed three times in phosphatebuffered saline/0.05% Tween 20. Streptavidin PE (100 ul, 4 ug/mL) isapplied and incubated for 30 minutes with shaking. Beads are washed onetime in phosphate buffered saline/Tween 20, resuspended in Luminexsheath fluid and analyzed on the Luminex system.

Dried blood-spot specimens from newborns are then tested following theabove protocol. For each specimen, both the hct and T₄ are measuredusing eluate from the same punch taken from the dried blood-spot.

Computer algorithms provide the calculated values for the concentrationof T₄ in each specimen, corrected for the hct. The algorithms use thedata obtained from the Hb calibration samples to estimate the hct valuefor each newborn dried blood-spot specimen, as described in Example 1,which is then used to provide a more accurate, hct-adjusted, T₄ serumconcentration value. Importantly, both the hct and the T₄ biomarker aremeasured from the same punch taken from the same dried blood spotspecimen.

Example 4

The following is a protocol for performing a multiplexed immunoassaymeasuring the five biomarkers T4 (thyroxine), TSH (thyrotropin), IRT1and IRT2 (immunoreactive trypsin isoforms 1 and 2), and 17OHP(17-hydroxyprogesterone) and using the Luminex 100 assay (from LuminexCorporation (Austin, Tex.)). Specific examples of the reagents listed inthe protocol are provided in following Example 5.

1. Multiplex Assay Protocol

Elute dried blood sample (DBS) standards overnight @23 degrees C., in ashaker @650 rpm in microtiter plate using 100 μL/well of Elution/AssayBuffer.

Transfer 75 μL of the eluates to pre-wet the wells of the filter plate.Add 25 μL of a mixture of T4 detector antibody and 17OHP tracer to eachwell.Add 25 μL of the Luminex bead mix to each well.Seal with plate sealer, cover, and shake at 37° for 3 hr.Aspirate, wash wells 3× with 150 μL Wash Buffer.Resuspend beads in 100 μL of a mixture of TSH detector and Trypsin 1 andTrypsin 2 detector antibodies. Cover, shake at 37° for 1 hr.Aspirate, wash wells 3× with 150 μL Wash Buffer.Resuspend beads in 100 μL of 4 μg/mL streptavidin-phycoerythrin (PJRS30,Prozyme).Cover, shake at 37° C. for 30 min.Aspirate, wash wells 3× with 150 μL Wash Buffer.Resuspend beads in 110 μL Sheath Fluid (Luminex cat# 40-50000).Analyze in Luminex 100 with sample size=80 μL, reading 100 beads of eachset, and gate setting=8000 to 13500.

2. Reagent Stocks

DBS Standards/Calibrators and Controls

CDC Multiplex Calibrators: CDC set #6

Microspheres (Beads)

Bead Mix: Beads were coupled according to protocol of manufacturer

(Luminex protocol.

Assay Buffer containing mix of beads conjugated to (a)Thyroxine(T4)-BSA, (b) mAb (monoclonal antibody) to Thyroid StimulatingHormone(TSH), (c) mAb to human Trypsin 1, (d) mAb to human Trypsin 2 and(e) polyclonal anti-17OHP:

-   -   (a) T4-BSA (Fitzgerald 80-IT50); conjugated to Luminex bead 137    -   (b) mAb to TSH (clone 204-12410) (Meridian (OEM) MAT04-410);        conjugated to Luminex bead 136    -   (c) mAb to human Trypsin 1 (clone 2C4) (Thermo Pierce Ab Shop        HYB 021-08-02); conjugated to Luminex bead 183    -   (d) mAb to human Trypsin 2 (Medix B607); conjugated to Luminex        bead 177    -   (e) polyclonal Ab to 17OHP (Ab 305, from private source);        conjugated to Luminex bead 121.

Detector/Reporter Antibodies and Tracer

T4 reporter: Biotinylated mAb to human Thyroxine (OEM ConceptsMAT02-525); 0.35 μg/mL)

-   TSH reporter: Biotinylated mAb to Thyroid Stimulating Hormone (clone    M94205, Fitzgerald 10-T25B); 4 μg/mL-   Trypsin 1+Trypsin 2 detector mix: Biotinylated rabbit anti-human    Trypsin (Biodesign K50900R), 5 μg/ml+Biotinylated anti human Trypsin    1/2/3 (R&D-   BAF3586), 1.25 μg/ml-   17OHP tracer: Biotinylated 17OHP; use at 1:64,000 dilution-   SAPE: Streptavidin-phycoerythrin (Prozyme PJRS30); 4 μg/mL    T4 reporter, TSH reporter, 17OHP tracer and rabbit anti-human    trypsin detector are biotinylated using Thermo Scientific Prod.    #21327 No-Weigh Sulfo-NHS-LC-Biotin. Biotinylation is performed    according to manufacturer's protocol.

Buffers

Elution/Assay Buffer: The assay buffer contains 9 g of NaCl, 0.5 g ofNaN₃, 10 g of BSA, 0.5 g of bovine globulin, 5 g of PEG6000, 0.1 mlTween 40, 0.5 mg of Danazol, 1 mg of Aprotinin per liter of 50 mMTris-HCl buffer, pH 7.75.

Wash Buffer: Tris-HCl buffered (7.8) salt solution with Tween 20

Example 5

A multiplexed immunoassay was performed to measure the five biomarkersT4 (thyroxine) and TSH (thyrotropin), IRT1 and IRT2 (immunoreactivetrypsin isoforms 1 and 2), and 17OHP (17-hydroxyprogesterone) in eluatesof dried blood samples, to assay for the conditions of congenitalhypothyroidism (CH), cystic fibrosis (CF), congenital adrenalhyperplasia (CAH). The assay was performed using the Luminex 100 assay(from Luminex Corporation (Austin, Tex.)). The dried blood samples wereobtained from the New York State Department of Health Newborn ScreeningLaboratory under Institutional Review board Protocol number 07-016. Noidentifying information was transferred with the specimens.

Multiplex Assay Procedure:

Multiplex standards for each biomarker were obtained from the Centersfor Disease Control (CDC). A 3 mm punch from each standard was elutedovernight at 23 C, in shaker at 650 rpm in a microtiter plate using 100μL/well of elution/assay buffer (containing 9 g of NaCl, 0.5 g of NaN₃,10 g of BSA, 0.5 g of bovine globulin, 5 g of PEG6000, 0.1 ml Tween 40,0.5 mg of Danazol, 1 mg of Aprotinin per liter of 50 mM Tris-HCl buffer,pH 7.75). A portion of the eluate (75 μL) was transferred to the wellsof a filter plate (which contained a built-in filter used to remove anyresidual paper fibers dislodged during elution) to pre-wet the wells.The eluate was then filtered prior to use in the assay.

The dried blood spots (a single 3 mm punch per well) were elutedovernight at room temperature in 100 μl of elution buffer with shaking.

To perform the multiplex assay, 75 μl of the sample eluate was combinedwith 25 μL of T4 detector (biotinylated mAb to human Thyroxine (OEMConcepts MAT02-525); 0.35 μg/mL) and 25 μL of the 17OHP tracer(biotinylated 17OHP; used at a 1:64,000 dilution). The Luminex bead mix(25 μL) was added (the bead mix consisted of the assay buffer containingLuminex beads conjugated to (i) thyroxine(T4)-BSA (Fitzgerald 80-IT50,conjugated to bead 137); (ii) mAb to thyroid stimulating hormone (TSH)(clone 204-12410, Meridian (OEM) MAT04-410, conjugated to bead 136));(iii) mAb to human Trypsin 1 (human Trypsin 1 (clone 2C4), Thermo PierceAb Shop HYB 021-08-02, conjugated to bead 183); and (iv) polyclonalanti-17OHP (polyclonal antibody (Ab) to 17OHP) (Ab 305, private source,conjugated to bead 121). The T4 reporter, TSH reporter, 17OHP tracer andrabbit anti-human trypsin detector were biotinylated using ThermoScientific Prod. #21327 No-Weigh Sulfo-NHS-LC-Biotin according to themanufacturer's protocol. The microtiter plate was then sealed with platesealer, covered and shaken at 37 C for 3 hour. Afterwards, each well wasaspirated and the beads were washed three times with 150 μL wash buffer(Tris-HCl buffered (pH 7.8) salt solution with Tween 20).

The beads were resuspended in 100 μL of TSH detector (biotinylated mAbto Thyroid Stimulating Hormone (clone M94205 (Fitzgerald 10-T25B), 4μg/mL), trypsin I and II detector antibody (biotinylated rabbitanti-human trypsin (Biodesign K50900R), 5 ug/ml; and biotinylatedanti-human trypsin 1/2/3 (R&D BAF3586), 1.25 ug/ml). The microtiterplate was then covered and shaken for 1 hour at 37 C, and the wells wereaspirated and washed three times with 150 μL Wash Buffer.

The beads were then resuspended in 100 μL of 4 μg/mL strep-avidinphycoerythrin (PJRS30, Prozyme). The microtiter plate was then coveredand shaken for 30 minutes at 37 C, after which the wells were aspiratedand washed three times with 150 μL Wash Buffer. The beads were againresuspended in 110 μL Sheath Fluid (Luminex cat# 40-50000) and analyzedin a Luminex 100 using a sample size of 80 μL, reading 100 beads of eachset, with a gate setting of 8000 to 13500.

Before the dried blood sample eluates were assayed for all fivebiomarkers in the multiplex assay, the assay protocol was validated foreach biomarker group/condition:

Validation of Individual Biomarker Assays Using the Multiplex AssayProtocol

Congenital Hypothyroidism (CH)

The target biomarkers for CH are thyroxin (T4) and thyrotropin (TSH).FIG. 4 presents the data obtained for the CH validation assay. Includedin FIG. 4 are standard curves for each biomarker. The results fromassaying control specimens and residual newborn spots are shown in FIGS.5 and 6. It is noted (see FIGS. 7 and 8) that there is close agreementbetween the performance of the multiplex assay when compared with thesetwo specimen types.

Cystic Fibrosis (CF)

The target biomarkers for CF are two isoforms of immunoreactive trypsin,trypsin 1 and trypsin 2. Data obtained for the CF validation assay areshown in FIGS. 9 and 10. Included in FIG. 9 are standard curves for eachbiomarker and results from assaying residual newborn spots (FIG. 10)compared with results obtained using the assay currently used in thescreening program. In FIG. 10, the performance of the combined IRT1/2bead sets is compared with results from the newborn screening program.As can be seen from FIG. 10, total trypsin (1+2) identified seven ofeight CF carriers. Use of the ratio of these two biomarkers identifiedseven of eight specimens as carriers. The carrier status in the newbornscreening program could only be determined by DNA analysis of the eightspecimens. FIG. 10 also demonstrates that there is close agreementbetween the performance of the multiplex assay when compared with thesetwo specimen types.

Congenital Adrenal Hyperplasia (CAH)

The target biomarker for CAH is 17-hydroxy-progesterone (17OHP), thesame as used routinely in newborn screening programs. FIGS. 11 and 12show the data obtained for the CAH validation assay. Included in FIG. 11is the standard curve for 17OHP and, in FIG. 12, the results fromassaying residual newborn spots compared with the currently used assayin the screening program. In FIG. 12, the values obtained using theLuminex assay (y axis) are plotted as a function of the correspondingvalues obtained using the currently used assay (x axis). As can be seenfrom FIG. 12, there is close agreement between the performance of themultiplex assay when compared with values from the screening program.

Multiplex Assay (CH/CF/CAH)

The multiplex assay described above was performed for a series of fourresidual newborn specimens using a single 3 mm punch taken from eachspecimen to test for the multiple biomarkers for CH (2 biomarkers), CF(2biomarkers), and CAH (1 biomarker). FIG. 13 presents a table showing theresults of the assay for each specimen, and the corresponding valuesobtained in the newborn screening program. It can be noted from FIG. 13that there is close agreement between the performance of the multiplexassay when compared with the values obtained individually in the currentnewborn screening program.

Determination of Hematocrit

Measurement of total hemoglobin can be used to calculate the hematocritof a specimen (Kokolm 1991; see FIG. 1). FIG. 14 shows the standardcurve used for measurement of total hemoglobin (Hbg's A, F, S, C) in adried blood specimen and the data derived from that curve used tocalculate the hematocrit of the newborn specimen samples used in themultiplex assay. Comparison of these values with the values obtained forthe same specimen using the Drabkins reagent show close comparability.

Example 6

The following describes a series of multiplex assays useful forscreening newborns for cystic fibrosis.

Newborn screening for cystic fibrosis (CF) has evolved following thereport in 1979 by Crossly et al. (Lancet 1:742-44 (1979)) that blood IRTlevels are higher in newborn infants with CF. There are severalmolecular forms of IRT, the two major forms secreted by exocrine cellsof the pancreas are trypsinogen 1 (cationic trypsinogen, IRT1) andtrypsinogen 2 (anionic trypsinogen, IRT2) (Guy, O, et al., Biochemistry17(9):1669-75 (1978); Kimland, M, et al., Clinica Chimica Acta 184:31-46(1989)). Normally the IRT1 form is present in higher levels, however inpathological conditions such as pancreatitis the IRT2 form becomespredominant (Itkonen, O, et al., J. Lab. Clin. Med. 115:712-8 (1990)).Today 46 states provide NBS for CF, all using IRT for the initialscreen. For the year 2007, the most recent year with complete data,9,076 infants were screen positive and 300 confirmed with CF, a ratio of30:1 screen positive to confirmed CF. (http://www2.uthscsa.edu/nnsis/).Subsequent testing after an initial screen positive can use a number ofdifferent protocols (Wilcken, B., J. Inherit. Metab. Dis. 30:537-543(2007)) in an effort to minimize the number of false positive results,such as IRT positives tested again on a newly collected specimen; IRTwith DNA analysis on that same first specimen, and others.

A number of investigators have developed immunoassays to IRT, andcommercial assays currently in use have employed both monoclonal andpolyclonal antibodies for IRT (Deam, S M, et al., Wein Klin Wochenschr100:55-7 (1988); Cabrini, G., et al., Clin. Biochem. 23:213-19 (1990);and Ball, C L, et al., Clin. Chem. Lab. Med. 43(5):570-572 (2005)). Theheterogeneous nature of IRT and differing specificity of antibodies tothe various components have raised issues with the standardization andexternal QC of the assay. As noted by Li et al (Li, L, et al., Journalof Medical Screening 13:79-84 (2006)), the lack of a universallyacceptable IRT standard has made the comparison of absolute IRT valuesamong commercial immunoassays difficult. As reported by Lafont (Lafont,P, et al., Clinica Chimica Acta 235:197-206 (1995)) trypsinogen existsin many forms in the serum. These different forms of trypsinogen are notrecognized equally among immunoassays, thus contribute to the discordantresults when comparing assay to assay.

In the present study we report development of a suspension arraymultiplexed immunoassay for the two specific isoforms of trypsinogenIRT1 and IRT2. The specificity of the assay for the two isoforms allowsdevelopment of external QC for the heterogeneous forms of IRT and allowsfor analysis of the IRT1:IRT2 ratio as a potential added parameterbefore referral for mutation analysis.

Materials and Methods Antibody Reagents

Anti-trypsin isoform specific monoclonal antibodies were coupled toLuminex xMAP microspheres following the manufacturers protocol. IRT1capture monoclonal antibody HYB 021-08-02 was obtained from AffinityBioreagents, the IRT2 capture monoclonal 8607 was obtained from MedixBiochemica. Polyclonal detector antibody K50900R (BiodesignInternational) was biotinylated using the Fluoreporter biotin-XXlabeling kit (Invitrogen) according to manufacturers instructions. Asheep polyclonal anti-trypsin 1/2/3 BAF3586 was purchased with thebiotin label from the manufacturer (R&D Systems). The two antibodieswere combined to make the detector mix, with K50900 at a concentrationof 5.0 ug/mL and BAF3586 at 1.25 ug/mL.

Assay Calibrators

IRT1 calibrators were used from the comparison reference method kit(MPBiomedical). IRT2 calibrators were prepared from recombinant IRT2(R&D Systems). Serum was treated with activated charcoal according tothe method of Li et al (Li, L, et al., Journal of Medical Screening13:79-84 (2006)) and combined with washed red blood cells to make wholeblood at 55% hematocrit. Aprotinin (Sigma Chemical) was added at aconcentration of 1 mg/L. The reconstituted whole blood was enriched withthe recombinant IRT2 and dispensed to make the dried blood spotcalibrators. The spots were air dried overnight, packed with desicantand stored frozen at −20 deg. C.

Assay Procedure

Assay buffer was prepared containing phosphate buffered saline (SigmaChemical Co.), 0.055 Tween 20, 0.05% sodium azide, 0.2% gelatin. 1 mg/Laprotinin (Sigma Chemical Co.) was added to the assay buffer to preparethe spot elution buffer. The dried blood spots (a single 3 mm punch perwell) were eluted overnight at room temperature in 100 ul of elutionbuffer with shaking. For the assay, 75 ul of the sample eluate wascombined with 25 ul of the trypsin 1 and trypsin 2 bead mix in order toobtain 2000 microspheres per well for each of the analytes. The captureincubation was for 3 hours at 37 deg. C. with shaking Microspheres werewashed three times in 100 ul assay buffer, and 100 ul of the antitrypsin detector antibody mix was added to each well. The detectorantibodies were incubated for 1 hour at 37 deg. C. with shaking, and themicrospheres were again washed three times with 100 ul assay buffer. Fordetection, 100 ul of streptavidin PE (Invitrogen, 5-866) was added at 4ug/mL, and incubated for 30 minutes at 37 deg. C. The assay plate wasaspirated and the microspheres resuspended in 100 ul of Luminex sheathfluid for analysis.

Samples

All clinical samples assayed were obtained from the New York State Dept.of Health Newborn Screening Laboratory under an Institutional ReviewBoard Protocol number 07-016. No identifying information was transferredwith the specimens.

Results Correlation

The selection criteria for the samples analyzed in the correlation studyare shown in TABLE 1:

TABLE 1 Correlation Study Sample Selection Criteria Reference IRT Samplevalue range ng/mL N IRT < 35 32 IRT 35-55 32 IRT 55-100 32 IRT 100-17040 IRT > 170 32IRT1+IRT2 (IRT1−IRT2) screen negative by the IRT1 and IRT2 assay werecompared with the IRT-R and had a correlation coefficient of 0.75 (seeFIG. 15), with a mean value lower than the IRT1, IRT1/IRT2: X=63.8+/−SD62.6, the IRTR: X=92.9+/−SD 69.0. This is not surprising due to theextremely different formats of the assays and different antibodies. (11)Of the 133 samples that were screen negative by the IRTR assay, 11 werescreen positive using the IRT1/IRT2 assay. Having no link back to thespecimen, it was impossible to retest and verify these findings. Of the32 cases screen positive by the IRTR, 11 cases were screen negative bythe IRT1 and IRT2 method. We have no way to show a screen negative orpositive by IRT2 alone. Each of these screen negative cases had beenconfirmed to have no CF mutations by the screening program in its secondtier mutation analysis.

Screen Positive Sample Evaluation

The screen positive sample population consisted of ten confirmedpositive cases with 2 CF mutations; six cases with two CF mutations,disease not yet confirmed; eight cases with 1 CF mutation; and, 137cases with no CF mutations detected. The screen positive cut-offestablished by the New York DOH NBS laboratory for the reference methodis a value greater than 170 ng/mL including the top 5% of each assay.The screen positive sample evaluation shown in TABLE 2 indicated that atotal trypsin (sum of IRT1 and IRT2) cut off >97 ng/mL would benecessary to achieve 100% sensitivity for the confirmed diseasepopulation. Calculation of the IRT1:IRT2 ratio for this populationindicated that a ratio <2.0 is also consistent with the elevated IRTRvalue.

TABLE 2 Screen Positive Sample Evaluation Refer- Trypsin Trypsin TotalTryp1/ ence Two Mutations 1 2 Trypsin Tryp2 IRT Confirmed Disease Ng/mLNg/mL ng/mL ng/mL ng/mL Del F508/3121 + G > A 121 129 250 0.938 248 DelF508/Del F508 63.6 93.2 156.8 0.682 183.5 Del F508/Del F508 117 184 3010.636 248 Del F508/R553X 104 269 373 0.387 248 Del F508/Del F508 96.6103 199.6 0.938 248 Del F508/W1282X 265 326 591 0.813 248 Del F508/DelF508 131 129 260 1.016 248 Del F508/N1303K 111 130 241 0.854 248 DelF508/Undetected 154 354 508 0.435 248 Del F508/R117H, 7T, 67.6 33.6101.2 2.012 194.3 9T, var pF508/pF508 95.4 119 214.4 0.802 248 DelpE60x/Del F508 40.1 57.1 97.2 0.702 248 p.S549/c387delA 76.8 107 183.80.718 248 pR117H/pD1152H, 107 364 471 0.294 226.5 7T, 7T Del pF508/DelpF508 78.4 62.4 140.8 1.256 226.5 pG85E/pF508del 252 884 1136 0.285226.5

TABLE 3 shows the analysis of eight single mutation CF carriers thatwere screen positive by the IRTR assay. Seven of these carriers wouldalso screen positive by use of the IRT1/IRT2 cut off of 97 ng/mL. In the137 cases that were screen positive by the IRTR assay with no CFmutations detected, 26 would be screen negative using the IRT1/IRT2 cutoff of >97 ng/mL, a reduction of 19% in the false positive rate in thisselected study population.

TABLE 3 Carriers (1 CF mutation) Screen Positive by IRTR Trypsin TrypsinTotal 1 2 Trypsin Tryp1/ One Mutation ng/mL ng/mL ng/mL Tryp2 referencepR553X 45.4 70.7 116.1 0.642 197.9 pF508del 159 289 448 0.550 226.5pD1152H 223 187 410 1.193 226.5 c3120 + 1G > A 254 790 1044 0.322 226.5pA455E 59 130 189 0.454 186.3 pF508del 87.4 111 198.4 0.787 213.7 c711 +1G > T 44.3 51 95.3 0.869 174.7 pF508del 57.5 70.1 127.6 0.820 226.5*cut up >97 ng/ml **cutoff >2.0

Screen negative samples with confirmed disease Analysis of three casesof confirmed disease with an IRTR value below the 170 ng/mL cut off isshown in TABLE 4. Two of the three cases would be screen positive usingthe IRT1/IRT2 assay criteria of total IRT.

TABLE 4 Confirmed disease with reference IRT <170 ng/mL ConfirmedTrypsin Trypsin Total Tryp1/ Ref Disease 1 2 Trypsin Tryp2 IRT 2mutations ng/mL ng/mL ng/mL ng/mL ng/mL W1282X/N1303K 55.2 49.8 1051.108 147.6 Del508/Del508 28.1 48.7 76.8 0.577 67.9 Del508/Del508 38.573.8 112.3 0.522 111.8

Population Study

A total of 597 population study samples were analyzed, the distributionis shown in FIG. 16. Two cases in this population were screen positiveby the IRT1-IRT2 criteria, of these one case fell within the top 5% ofthe reference IRT method and had 1 CF mutation detected, the second casewas screen negative by the reference IRT.

The screen false positive rate in newborn screening for CF has remainedpersistently high, despite numerous attempts to lower it. (Wilcken, B.,J. Inherit. Metab. Dis. 30:537-543 (2007)) One unexplored approach, useof the two isoforms of trypsin, was examined in these studies. The goalin this study was the development of a multiplexed assay for CF usingthe two major trypsinogen isoforms that would meet screening standardsfor clinical accuracy in comparison to current commercial IRT assays.

The correlation study showed substantially equivalent performance of theassays in segregation of a screen positive population. Importantly forthe 11 discrepant cases that were screen positive in the IRTR but screennegative in the IRT1/IRT2 assay, no CF mutations were detected in themby the screening program as part of its protocol, suggesting a greatersensitivity for the multiplex assay. Analysis of a screen positivepopulation with confirmed disease indicated that a cut off of >97 ng/mLin the IRT1/IRT2 assay would be needed to achieve 100% sensitivity forthese samples. Although this cut off is substantially lower than the onedeveloped for the reference method of 170 ng/mL, it is nearer to one of112 ng/mL reported for a monoclonal antibody based method for total IRT.(Ball, C L, et al., Clin. Chem. Lab. Med. 43(5):570-572 (2005)). Li, etal. (Li, L, et al., Journal of Medical Screening 13:79-84 (2006))reported that the measured immunoreactivity of an IRT spiked preparationwas 45%-60% of that specified by the company providing the material. Itis possible that more specific immunoreactivity is observed whenmeasuring the two isoforms separately as reported here.

Cystic fibrosis carriers have been shown to have higher IRT values thanthe normal population (Casellani, C, et al., Am. J. Med. Genet. A135(2):142-4 (2005); Lecoq, I, et al., Acta Paediatr. 88:338-341(1999)). In a screening program in which the goal is detect disease andnot carrier status, correct identification of carrier status could be agreat help. In these studies use of the total IRT1-IRT2 was unable todiscriminate the carrier population, with seven of eight carriers screenpositive by the reference assay also screen positive by the IRT1/IRT2criteria.

In three cases identified with confirmed disease that had a referenceIRT below the cut off, two were screen positive by the IRT1/IRT2criteria. More studies are needed to determine whether these resultsindicate that the IRT1/IRT2 assay has greater specificity.

This study demonstrates that the IRT1/IRT2 multiplexed assay for CF hassubstantial equivalence in detecting screen positive specimens comparedwith the reference IRT method. The specificity of the antibodies for thetwo isoforms would also provide advantages in the standardization andexternal QC of the assay. Perhaps more importantly, the multiplex formatwill allow additional biomarker e.g., PAP (Sarles, J, et al., J.Pediatr. 147:302-5 (2005)), to be added in the future. An even moreoptimistic goal is the combining of this CF assay with immunoassays forcongenital hypothyroidism, and congenital adrenal hyperplasia into asingle assay for the three, thereby saving time in a screeninglaboratory specimen usage, and perhaps at a lower cost.

All publications cited in this specification are herein incorporated byreference in their entirety to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference in its entirety. While the invention has been describedwith reference to a particularly preferred embodiment, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A method for determining a concentration of a biomarker in the serumof a patient, said method comprising: (a) obtaining a blood sample froma patient; (b) obtaining an extract from the blood sample; (c) measuringa concentration of hemoglobin (Hb) in the extract; (d) calculating ahematocrit (hct) value for the blood sample from the measuredconcentration of Hb; (e) measuring a concentration of a biomarker in theextract; and (f) determining a concentration of the biomarker in theserum of the patient using the calculated het value, volume of the bloodsample, and the measured concentration of biomarker in the extract. 2.The method of claim 1, wherein the blood sample is a dried blood sample.3. The method of claim 1, wherein a computer algorithm is used todetermine the concentration of the biomarker in the serum of the patientusing the calculated hct value, volume of the blood sample, and themeasured concentration of biomarker in the extract.
 4. The method ofclaim 1, wherein the biomarker is a metabolic biomarker.
 5. The methodof claim 4, wherein the metabolic biomarker is selected from the groupconsisting of T4, TSH, and an amino acid.
 6. The method of claim 1,further comprising (g) measuring a concentration of a second biomarkerin the extract and determining a concentration of the second biomarkerin the scrum of the patient using the calculated hct value, volume ofthe blood sample, and the measured concentration of the second biomarkerin the extract.
 7. The method of claim 4, wherein the metabolicbiomarker is suitable for use in a newborn screening program.
 8. Themethod of claim 2, wherein the patient is a human infant.
 9. The methodof claim 1, wherein step (c) is conducted using an affinity-based assay.10. The method of claim 9, wherein the affinity-based assay comprises animmunoassay.
 11. The method of claim 1, wherein step (e) is conductedusing an affinity-based assay.
 12. The method of claim 11, wherein theaffinity-based assay comprises an immunoassay.
 13. The method of claim1, wherein steps (c) and (e) are conducted using a multiplexedimmunoassay.
 14. The method of claim 1, further comprising identifyingwhether said patient possesses an aberrant hct level.
 15. The method ofclaim 14, wherein said aberrant hct level is indicative of anemia.
 16. Amethod of calculating a concentration of a biomarker in the serum of apatient, the method comprising: (a) measuring a concentration of Hb anda concentration of a biomarker in an extract obtained from a dried bloodsample from a patient; (b) converting the measured concentration of Hbto a hct value; and (c) calculating a concentration of the biomarker inthe serum of the patient using the hct value and the measuredconcentration of the biomarker in the extract.
 17. A method of arrivingat concentrations of one or more biomarkers in a patient's whole blood,the method comprising: (a) obtaining a punch from a dried whole bloodspecimen of a patient, wherein the dried whole blood specimen is madefrom a whole blood sample taken from the patient; (b) obtaining aneluent from the punch; (c) measuring concentrations of one or morebiomarkers and of Hb in the eluent or a diluent thereof; (d) estimatingan het fraction of the whole blood sample from the measuredconcentration of Hb; and (e) adjusting the measured concentrations ofthe one or more biomarkers based on the estimated hct fraction to arriveat concentrations of the one or more biomarkers in the serum fraction ofthe patient's whole blood.
 18. The method of claim 17, wherein themeasuring step is conducted using a multiplexed assay system, whichmeasures substantially simultaneously the concentrations of at least theone or more biomarkers.
 19. A method of arriving at concentrations ofone or more biomarkers in a patient's whole blood using measurementsobtained from a dried whole blood specimen, the method comprising: (a)measuring concentrations of one or more biomarkers in an eluent from apunch of a dried whole blood specimen made from a whole blood sampletaken from a patient; (b) measuring, a concentration of Hb in the eluentor a diluent thereof; (c) estimating an hct fraction for the whole bloodsample from the measured concentration of Hb; and (d) adjusting themeasured concentrations of the one or more biomarkers based on theestimated hct fraction to arrive at concentrations of the one or morebiomarkers in the patient's whole blood.
 20. A method for determining aconcentration of Hb in a sample, said method comprising: (a) contactingthe sample with a fluorogenic substrate for peroxidase and hydrogenperoxide, such that when Hb is present a fluorescent product isproduced; (b) determining the amount of the fluorescent product; and (e)calculating the concentration of the Hb in the sample based upon theamount of the fluorescent product determined in (b).
 21. The method ofclaim 20, wherein the sample is an extract of a blood sample obtainedfrom a patient.
 22. The method of claim 20, wherein the substrate isAMPLEXRED or AMPLEX ULTRARED.
 23. The method of claim 21, furthercomprising (a) calculating a hct value for the blood sample using theconcentration of the Hb in the extract; (b) measuring a concentration ofa biomarker in the extract; and (c) determining a concentration of thebiomarker in plasma of the blood sample from the patient using thecalculated hct value, volume of the blood sample, and the concentrationof the biomarker in the extract. 24-27. (canceled)
 28. An assay bufferfor the substantially simultaneous analysis of two or more biomarkers,the buffer comprising, in an aqueous mixture: (i)tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl); (ii) sodiumchloride (NaCl); (iii) one or more emulsifiers; (iv) bovine serumalbumin (BSA); (v) polyethylene glycol (PEG); (vi) one or morepreservatives; (vii) bovine globulin; and (viii) a testosteronederivative. 29-42. (canceled)
 43. An aqueous buffer for thesubstantially simultaneous analysis of two or more biomarkers, thebuffer comprising, in an aqueous mixture: (i) about 50 mM Tris-HCl; (ii)about 150 mM NaCl; (iii) about 0.02% Tween 40; (iv) about 1% BSA; (v)about 0.5% polyethylene glycol; (vi) a preservative; (vii) about 0.05%bovine globulin; (viii) about 0.5 mg/L danazol; and (ix) about 1 mg/L ofa protease inhibitor; provided that the assay buffer does not includeabout 0.5 mg or more per liter of 8-anilino-1-naphthalenesulfonic acidor a salt thereof. 44-51. (canceled)
 52. A method of arriving atconcentrations of two or more biomarkers in a serum portion of a wholeblood sample taken from a patient, the method comprising: (a) obtainingone or more punches from a dried whole blood specimen of a patient, inwhich the dried whole blood specimen is made from a whole blood sampletaken from the patient; (b) obtaining an eluent from the one or morepunches using a universal buffer as an elution solvent; (c) measuring aconcentration of each of two or more biomarkers and of hemoglobin (Hb)in the eluent or a diluent thereof, provided that the measurement of theconcentration of each of the two or more biomarkers is carried outsubstantially simultaneously using a multiplexed affinity assay; (d)estimating a hematocrit (het) value for the whole blood sample from theconcentration of Hb measured in step (c); (e) using the estimated hetvalue to adjust the concentration of each of the two or more biomarkersmeasured in step (c) to arrive at concentrations of two or morebiomarkers in a serum portion of the whole blood sample taken from thepatient. 53-71. (canceled)
 72. A method of determining whether a patienthas, or may develop, two or more disorders by using a multiplexedaffinity assay to determine the concentrations of two or more biomarkersindicative of the disorders in a serum portion of a whole blood sampletaken from the patient, the method comprising: (a) obtaining a punchfrom a dried whole blood specimen of a patient, in which the dried wholeblood specimen is made from a whole blood sample taken from the patient;(b) obtaining an eluent from the punch using a universal buffer as anelution solvent; (c) measuring a concentration of each of two or morebiomarkers and of total hemoglobin (Hb) in the eluent or a diluentthereof, provided that the measurement of the concentration of each ofthe two or more biomarkers is carried out substantially simultaneouslyusing a multiplexed affinity assay; (d) estimating a hematocrit (hct)value for the whole blood sample from the concentration of total Hbmeasured in step (c); (e) using the estimated het value to adjust theconcentration of each of the two or more biomarkers measured in step (c)to arrive at adjusted concentrations of the two or more biomarkers in aserum portion of the whole blood sample taken from the patient; and (f)using the adjusted concentrations of the two or more biomarkers todetermine whether the patient has, or may develop, two or moredisorders. 73-78. (canceled)